Novel approach to molecular diagnosis of human papillomavirus-related diseases

ABSTRACT

The present invention relates to an accurate, sensitive, and efficient sequential or concurrently sequential method for molecular diagnosis of human papillomavirus (HPV)-based disease, where the method improves the accuracy and reliability of diagnostic and prognostic assessments of HPV-based disease. The method of the invention comprises a primary screen of a sample for HPV nucleic acids, followed by a secondary screen for molecular markers, such as proliferation and cell cycle control group protein markers. The sequential or concurrently sequential method significantly reduces the number of false positive results.

This application is a continuation application under 35 U.S.C. §120 ofU.S. patent application Ser. No. 10/411,830, filed Apr. 11, 2003,entitled “Novel Approach To Molecular Diagnosis Of HumanPapillomavirus-Related Diseases” which is incorporated herein byreference as if fully set forth herein.

FIELD OF THE INVENTION

The present invention is generally related to the field of molecularassays and specifically to the area of assays for the assessment ofdisease using a sensitive, accurate assay for diagnosis and prognosis ofhuman papillomavirus (HPV)-related diseases.

BACKGROUND OF THE INVENTION

The detection and diagnosis of disease is of obvious importance for thetreatment of disease. Numerous characteristics of diseases have beenidentified and many are used for the diagnosis and prognosis of disease.Many diseases are preceded by, and are characterized by, changes in thestate of the affected cells. Changes may include the expression of viralgenes in infected cells, changes in the expression patterns of genes inaffected cells, changes in enzymatic activities, and changes in cellmorphology. The detection, diagnosis, and monitoring of diseases may beaided by the assessment of such cell states, especially by improving theaccuracy of detection.

The worldwide annual incidence of cervical cancer is approximately500,000 cases^(1,2). The current diagnostic assay for early cervicalcancer detection is the Papanicolaou (Pap) test which is a simplemorphological screening method of examining strained exfoliative cells.It is used most commonly to detect cancers of the cervix, but it may beused for tissue specimens from any organ. The findings are usuallyreported descriptively and grouped into several classifications,including the Papanicolaou and the cervical intraepithelial neoplasia(CIN) classifications.

The Pap test contributed to a 74% decline in deaths due to cervicalcancer in the United States between 1955 and 1992³. However, thesensitivity of the Pap test for high-grade cervical lesions (cervicalintraepithelial neoplasia grade 3 or CIN 3) is not very high, typicallyranging from as low as 50% for conventional Pap tests to 85% for thenewer liquid cytology tests⁴. Routine use of advanced cytology-basedautomated reading technologies such as Auto-Pap® 300 QC. AUTOCyt®, andCyto-Savant™ is not a favorable approach due to high cost⁵ and onlyminor increases in sensitivity for CIN 3⁶. The clinical data suggestthat HPV DNA tests are more sensitive (about 90 to 95%) than either theconventional (50 to 70%) or liquid-based (60 to 85%) Pap tests.Combination of the Pap test with the HPV DNA test reduces false-negativecases and produces combined sensitivity for CIN 3 at 95% to 100%.However, false negative cytology tests account for at least 25% ofinvasive cancer cases in the United States⁷. Additionally,false-positive cases are another important limitation of the cytologyapproach.

Alternative diagnostic approaches are to couple the Pap test with a testfor either HPV DNA^(8,9,10) or cellular molecular markers^(11,12,13).The combination of the Pap test with a HPV DNA test achieves highsensitivity and can partially address the relatively high rate of HPVpositive cases in women without prevalent cervical neoplastic disease.There is an increasing acceptance for those who apply appropriatealgorithms in recommending women who are positive for HPV alone to makefollow up appointments with longer intervals compared to those women whoare positive for both the HPV nucleic acid test and the Pap test who arerecommended to have immediate attention. The use of these algorithms andrecommendations will be of clinical benefit. High-risk HPV DNA is highlyassociated with cervical cancer¹⁴, however, most HPV infections do notlead to cervical cancer. It is estimated that 5% to 10% of normal womenare HPV infected by carcinogenic HPV types, and of these up toone-quarter may be expected to develop high grade cervical lesions¹⁵.Women over 30 years of age are more likely to develop neoplastic diseasethan younger women¹⁶.

Two formal classification systems are utilized for identification ofcervical cancer precursor conditions. The CIN system relates to thetissue biopsies classification and comprises of Negative; mild cervicaldysplasia or CIN 1; moderate dysplasia or CIN 2; severe dysplasia(including carcinoma in situ) or CIN 3; and carcinoma. The BethesdaClassification system relates to Pap changes and comprises of withinnormal limits (WNL) and benign cellular changes (equivalent toNegative); atypical squamous cell of undetermined significance (ASCUS)or atypical glandular cell of undetermined significance (AGCUS) favorbenign (no equivalent in the CIN system); ASCUS or AGCUS favor dysplasia(no equivalent in the CIN system); low grade squamous intraepitheliallesion (LSIL) (equivalent to CIN 1); high grade squamous intraepitheliallesion (HSIL) (equivalent to CIN 2-3); and carcinoma. (PATH Outlook Vol18@, # 1, 2000; Jelovsek FR Woman's Diagnostic Cyber).

Human papillomavirus (HPV) induces benign epithelial proliferations ofthe skin and mucosa in humans and is associated with anogenitalneoplasias and carcinomas. Human papillomaviruses characterized to dateare associated with lesions confined to the epithelial layers of skin,or oral, pharyngeal, respiratory, and, most importantly, anogenitalmucosae. HPV is a virus that is the cause of common warts of the handsand feet, as well as lesions of the mucous membranes of the oral, anal,and genital cavities. The virus may be transmitted through sexualcontact and is a precursor to cancer of the cervix. Non-limitingexamples of diseases, disorders, and conditions associated with HPVinclude cervical intraepithelial neoplasia (CIN), and cancer, cervicaldysplasia, vaginal cancer, vaginal dysplasia, vulvar cancer, penilecancer, anal cancer, oral cancer, atypical squamous cells includingatypical squamous cells of undetermined significance (ASCUS) andatypical squamous cells, high-grade squamous intraepithelial lesion(HSIL), genital warts, plantar warts, butcher's warts, and flat warts,condylomata, epidermo dysplasia verruciformis and other skin diseases,laryngeal papilloma, oral papilloma and conjunctival papilloma. Morethan 70 types of HPV have been identified, many of which have beenisolated from anogenital lesions.

HPV types may be divided into three groups according to their biologicaloncogenic potential, where some are associated with cancerous andpre-cancerous conditions. Low-risk HPV types are more frequentlyassociated with low grade squamous intraepithelial lesions (LSIL;suspect CIN 1 lesions) and condyloma acuminatum. This group comprises,but is not limited to, low-risk HPV types 6, 11, 42, 43, 44, and others.Intermediate-risk HPV types comprise, but are not limited to HPV types31, 33, 35, 39, 51, 52, 56, 58, 59, and 68. High-risk HPV typescomprise, but are not limited to HPV types 16, 18, and 45. High-risk HPVtypes are more frequently associated with high-grade squamousintraepithelial lesions (HSIL; suspect CIN 2, CIN 3) and invasivecarcinoma of uterine cervix.

The viral genome may be divided into three regions: (1) the upstreamregulatory region (URR) or long control region (LCR), containing controlsequences for HPV replication and gene expression; (2) the viral earlygene region, encoding, among others, the E2, E6 and E7 genes; and (3)the late region, encoding the L1 and L2 genes.

HPV gene expression in high-grade premalignant disease or cancer appearsrestricted to the early genes, possibly due to cellular differentiationarrest induced by the viral E6 and E7 genes. In comparison to active HPVinfection, E6 and E7 gene control in cancer is deranged by mutations inthe viral URR and, in integrated viral fragments, by the disruption ofthe viral E2 gene, stabilization of E6 and E7 mRNAs, and influences atthe cellular integration site. Cervical cells containingextrachromosomal HPV genomes rapidly segregate and are outgrown inculture by cells that contain integrated viral genomes¹⁷.

Cervical neoplastic progression and cancer are highly associated withHPV persistence and viral DNA integration in the cellular genome. Theseprimary molecular events, characteristic for high-risk HPV DNA subtypes,result in over-expression of the viral oncogenes E6 and E7. The presenceof high-risk HPV E6 and E7 proteins leads to inactivation of theimportant tumor-suppression proteins p53 and pRB and their associatedpathways¹⁸. As a result, p53- and pRB-associated genes and theirproducts (mRNA and protein) are aberrantly expressed. Some aberrantlyexpressed gene products are called markers due to their readilydetectability and association with neoplastic progressions. Thus,another alternative approach to improve sensitivity and/or specificityof the Pap test is to combine with marker-based screening. However, thecombination of Pap test with molecular markers will also have to accountfor false negatives and false positives due to limited markersensitivity and specificity (without information on high-risk HPV DNA)for neoplastic progression¹³. The published studies show highsensitivity of the molecular markers for high grade squamousintraepithelial lesion (HSIL) specimens. However, a high rate of certainmarker-positive cases (approximately 12%-26%) for the within normallimits (WNL) group, (approximately 74%-88%) for atypical squamous cellsof undetermined significance (ASCUS) and low grade squamousintraepithelial lesion (LSIL) groups have also beendemonstrated^(11,20,21,23). A high-rate of false positive cases is thusevident for some marker and Pap test combinations.

Several methods have been used to diagnose clinical or subclinicalinfection with HPVs including clinical observation, cytologicalscreening by Pap test, electron microscopy, immunocytochemistry, and DNAhybridizations. However, a definitive diagnosis of HPV infection dependson the detection of nucleic acids (DNA or RNA) or proteins of the virus.The detection of HPV DNA by nucleic-acid-based assays may identify20-30% of women with cervical disease who have false-negative results byPap test screening. The presence of HPV DNA in the cervical lesions ofolder women has a higher predictive value for the progression ofcervical intraepithelial neoplasia to invasive cancer than in youngerwomen. Thus, there is a need for a more accurate, sensitive, andefficient method of screening clinical specimens for neoplastic diseasethan the time consuming and subjective Pap cytology test screenings.There is also a need for further improvements to HPV nucleic acidtesting alone or the combination of Pap test screening and either HPVDNA or molecular marker tests.

Accordingly, it is an object of the present invention to provide anaccurate, sensitive, and efficient method for molecular diagnosis andprognosis of HPV-based disease, where the method as a whole incorporatesadded specificity, enabling individual steps of the method to use lowerstringency.

It is another object of the present invention to provide an assay toimprove the accuracy and reliability of diagnostic and prognosticassessments of HPV-based disease.

It is a further object of the present invention to provide a method forassessing the risk that a patient infected with HPV will have or willdevelop HPV-based high grade neoplastic disease.

Yet a further object of the present invention is to provide a method formonitoring the effectiveness of treatment of HPV-based disease.

Another object of the present invention is to provide a scaleidentifying the degree of HPV high risk disease progression.

A further object of the present invention to provide kits fordiagnosing, prognosing, and assessing the stage of HPV-based disease.

SUMMARY OF THE INVENTION

The present invention relates to a novel method for molecular diagnosisand prognosis of human papillomavirus (HPV)-related diseases, disorders,and conditions. This method relates to a sequential or concurrentlysequential method comprising (1) an HPV nucleic acid test, preferably anHPV DNA or HPV RNA test; followed by (2) a test for molecular markers,preferably specific protein markers. This novel approach will enable thedistinction of progressive HPV-related neoplastic disease from benignand non-progressive lesions. Additionally, this combination of testsperformed sequentially or concurrently sequentially acts as a moreaccurate and sensitive tool for diagnosis and staging of cancer,specifically cervical cancer, by significantly reducing the number offalse positive results. The number of false positive results issignificantly reduced by employing both the HPV nucleic acid test andthe test for specific protein markers, such as but not limited to cellproliferation and cell cycle control group proteins. The presentinvention may be used to assess the stage or risk of a disease asindicated by the state of the cells. It may also be used to guide orassess the effectiveness of a therapy for a disease by identifyingappropriate therapy based on the indicated cell state or by indicatingany change in the state of cells subjected to the therapy.

DESCRIPTION OF THE INVENTION

The present invention relates to the identification and monitoring ofhuman papillomavirus (HPV)-infected cells. One embodiment of the presentinvention relates to a method of measuring the presence and levels ofexpression of genes involved in a disease state, and comparing theirexpression to each other or to reference genes, as an indication of thestate of the cells. Such measurements are combined with a molecularmarker assay to increase the accuracy and reliability of the assessmentof the disease state by significantly reducing the number of falsepositive results. This sequential or concurrently sequential methodcomprising an HPV nucleic acid test and a molecular marker assay, suchas protein marker, incorporates specificity in diagnosing and prognosingHPV infected individuals.

One advantage of the specificity being incorporated in the sequential orconcurrently sequential method is that the individual steps may uselower stringency conditions. For example, the HPV nucleic acid test ofthe sequential or concurrently sequential method may be less stringentas compared to the results of the HPV nucleic acid test alone. Althoughthis step of the method is less restrictive, overall the sequential orconcurrently sequential method removes cross reactivity sincespecificity is incorporated. This method may also be used to guide orassess the effectiveness of a therapy for a disease by identifying theappropriate therapy based on the indicated disease state or byindicating any change in the state of cells subjected to the therapy.The present invention is generally directed to a sequential orconcurrently sequential method comprising measuring gene or viral genomepresence or expression followed by detecting particular markersindicative of disease state. “Sequential” is defined herein as relatingto or arranged in a sequence, or following in order, or in regularsuccession without gaps. “Concurrently sequential” is defined herein asrelating to separate processes of separate samples, where the samplesare the same representative group, and the processes may be performed atthe same time. Whereas, “simultaneous” is defined herein as processes ofthe same sample set, where the processes occur, happen, or exist at thesame time.

A further embodiment of the present invention implements an accurate andsensitive sequential or concurrently sequential method of detecting andassessing disease and the stage of disease progression in a biologicalsample from a subject by measuring HPV nucleic acid infection, HPV DNAor HPV RNA, followed by detecting specific molecular markers. Inparticular, this embodiment relates to a protein-based moleculardetection method for detecting protein markers, such as cellproliferation and cell cycle control markers. This novel sequential orconcurrently sequential method is expected to be more specific inidentifying women who are more likely to have high-grade cervicaldisease among those who are infected by carcinogenic HPV DNA types orcorresponding HPV RNA. The test may also have utility in detectingpotentially progressive HPV infections in women who do not haveprevalent high-grade cervical neoplastic disease. This novel moleculardiagnostic method is a sequential or concurrently sequential method thatutilizes HPV nucleic acid detection as the primary screening testfollowed by protein marker detection as a secondary screening test,where the protein markers are cell proliferation and cell cycle controlgroup proteins.

Many diseases are characterized by specific cellular phenotypes and geneexpression patterns. For example, neoplastic and cancerous cellsgenerally exhibit certain distinctive morphologies and growthcharacteristics. Molecular characteristics, such as gene mutations andgene expression patterns are also a good indicator of diseaseprogression. Virally infected cells may exhibit different morphologiesand cellular gene expression patterns, including expression of viralgenes. In the present invention, the characteristics of the cell state,such as changes in the presence and expression of genes, including viralgenes, may be used to determine or assess the human papillomavirusdisease state from a patient sample.

The characteristics to be detected are generally specific to the cellstate of interest and the disease suspected of being present in the cellsample. Such characteristics may generally be divided into two types,cytological characteristics and molecular characteristics. As describedherein, cytological characteristics are characteristics such as, forexample, overall cell shape, nuclear shape, nuclear size, andappearance. The primary identification and classification of manyneoplastic and cancerous cells have traditionally been accomplished byusing cytological characteristics. Identification of cytologicalcharacteristics is generally slow and tedious, and requires a relativelyhigh level of training, and generally cannot be easily automated. Asused herein, molecular characteristics are determined by the presenceand state of particular molecular species, such as proteins, nucleicacids, and metabolites. Such molecular characteristics are generallyidentified by detecting and measuring the particular molecules ofinterest.

In one embodiment of the present invention, a method for the diagnosisand prognosis of HPV infection comprises a primary screen for detectingHPV nucleic acids by hybridization with DNA or RNA probes directedagainst specific types of HPV, such as those relating to high risk HPV.The probes are type-specific and may be labeled or unlabeled. If theprobe is labeled, the label may be isotopic or non-isotopic, preferablynon-isotopic; however, the preferred probe is not labeled or modified.Although all of the hybridization methods are highly sensitive andspecific, each has certain specifications associated with the timeneeded, the expertise, or the sample used in the procedure, but most ofall their sensitivity. Several different HPV hybridization methodologiesmay be used including, but not limited to, Southern blot, Dot blot, Slotblot, and in situ hybridization. Other non-limiting examples oftechniques for detecting HPV nucleic acids include, branched DNA assays,transcription-mediated amplification (TMA), ligase chain reaction (LCR),self-sustained sequence replication (3SR), nucleic acid sequence basedamplification (NASBA), strand displacement amplification, andamplification with Qβ replicase, and polymerase chaing reaction (PCR)including both low stringency (broadly cross-reactive) and highstringency (type-specific) methods. PCR-based methods have been usedsuccessfully for the detection and typing of genital HPV genotypes inclinical specimens, such as cervical swabs or scrapes, salinecervicovaginal ravages, frozen biopsies, and formalin-fixedparaffin-embedded tissues.

Numerous assays for the detection and measurement of gene expressionproducts are known and may be adapted for the determination of the levelof expression of genes of interest in the disclosed assay. For example,many of the techniques for the detection of HPV in general or expressionof HPV genes described below may also be adapted for use in thedisclosed assay for the detection of expression of HPV genes E6, E7, L1,E4, and E2. However, a preferred method of detecting HPV nucleic acidsin a cell sample is by the hybrid capture technique described in WO93/10263 by Digene, incorporated herein by reference.

Yet another embodiment of the present invention relates to the level ofstringency in the HPV nucleic acid test. Since the sequential orconcurrently sequential method of diagnosing and prognosing individualsinfected with HPV incorporates specificity, the HPV nucleic acid testmay be modified to use lower stringency conditions than when the HPVnucleic acid test is used alone. As will be understood by those skilledin the art, the stringency of hybridization may be altered in order toidentify or detect identical or related polynucleotide sequences. Aswill be further appreciated by the skilled practitioner, the meltingtemperature, T_(m), can be approximated by the formulas as known in theart, depending on a number of parameters, such as the length of thehybrid or probe in number of nucleotides, or hybridization bufferingredients and conditions (see, for example, T. Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1982 and J. Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989; Current Protocols in Molecular Biology, Eds. F. M.Ausubel et al., Vol. 1, “Preparation and Analysis of DNA”, John Wileyand Sons, Inc., 1994-1995, Suppls. 26, 29, 35 and 42; pp.2.10.7-2.10.16; G. M. Wahl and S. L. Berger (1987; Methods Enzymol.152:399-407); and A. R. Kimmel, 1987; Methods of Enzymol. 152:507-511).

As a general guide, T_(m) decreases approximately 1° C.-1.5° C. withevery 1% decrease in sequence homology. Also, in general, the stabilityof a hybrid is a function of sodium ion concentration and temperature.The hybridization reaction of the HPV nucleic acid test alone isperformed under conditions of high stringency. However, since thesequential or concurrently sequential method of the present inventionincorporates specificity and reduces both analytical and clinicalfalse-positive results, the primary HPV nucleic acid screen may uselower stringency conditions. Reference to hybridization stringency,e.g., high, moderate, or low stringency, typically relates to suchwashing conditions.

Thus, by way of non-limiting example, “high stringency” refers toconditions that permit hybridization of those nucleic acid sequencesthat form stable hybrids in 0.018M NaCl at about 65° C. (i.e., if ahybrid is not stable in 0.018M NaCl at about 65° C., it will not bestable under high stringency conditions). High stringency conditions canbe provided, for instance, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE (saline sodium phosphate EDTA) (1×SSPEbuffer comprises 0.15 M NaCl, 10 mM Na₂HPO₄, 1 mM EDTA), (or 1×SSCbuffer containing 150 mM NaCl, 15 mM Na 3 citrate 2H₂O, pH 7.0), 0.2%SDS at about 42° C., followed by washing in 1×SSPE (or saline sodiumcitrate, SSC) and 0.1% SDS at a temperature of at least about 42° C.,preferably about 55° C., more preferably about 65° C.

“Moderate stringency” refers, by non-limiting example, to conditionsthat permit hybridization in 30%-40% formamide, 5× Denhardt's solution,5×SSPE (or SSC), 0.2% SDS at 42° C. (to about 50° C.), followed bywashing in 0.2×SSPE (or SSC) and 0.2% SDS at a temperature of at leastabout 42° C., preferably about 55° C., more preferably about 65° C.

“Low stringency” refers, by non-limiting example, to conditions thatpermit hybridization in 10% formamide, 5× Denhardt's solution, 6×SSPE(or SSC), 0.2% SDS at 42° C., followed by washing in 1×SSPE (or SSC) and0.2% SDS at a temperature of about 45° C., preferably about 50° C.

For additional stringency conditions, see T. Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982). It is to be understood that the low, moderate andhigh stringency hybridization/washing conditions may be varied using avariety of ingredients, buffers and temperatures well known to andpracticed by the skilled artisan.

Another embodiment of the present invention is detection and measurementof the expression levels of certain HPV genes. An impressive amount ofdata has been accumulated over the years showing that carcinoma of thecervix is associated with infection of certain types of HPV. Though thepresence of HPV DNA in a precancerous lesion is indicative of anincreased relative risk for cervical dysplasia and invasive carcinoma,it is still difficult to predict the clinical behavior of precancerouscervical lesions. Tumors arise due to the accumulation of geneticalterations which may activate oncogenes and/or inactivate tumorsuppressor genes and/or genes involved in DNA damage recognition andrepair.

HPV E6 and E7 proteins are two oncoproteins of high-risk HPV types(e.g., HPV-16, HPV-18), which inactivate p53 and bind or inactivate pRbrespectively, and thus act as promoters of HPV-associated cancers. Theexpression levels of E6 and E7 oncoproteins encoded by high-risk HPVtypes are a sensitive and accurate measure of potential risk of an HPVinfection developing into a cancerous lesion. In one embodiment of thepresent invention, the HPV DNA or RNA test may be assayed by measuringrelative amounts of E6 and/or E7 expression levels and E2 and/or L1 inan HPV-infected lesion to determine the ratio of E6 and/or E7 to L1and/or E2, where this ratio is a direct measure of risk andsusceptibility to the development of a cancerous lesion, as described inU.S. Pat. No. 6,355,424 to Digene, incorporated herein by reference.

As can be readily discerned, each major disease state is represented bya unique expression pattern of these four genes, i.e., E6, E7, L1, andE2. Conditions expressing these genes are regarded as indicative of anincreased risk of potentially serious medical aliments. Otherrelationships involving the relative level expression of other HPV genes(such as E1, E4, E5, and L2), and other, non-HPV genes, may also be usedto assess cell state. For example, L2 and E4 are frequently associatedwith benign viral production diseases, and E1 is similar in profile toE2 and is often deleted in malignancies. Other relationships ofexpression for these HPV proteins may exist for other HPV-baseddiseases, and the disclosed method may be used to assess the state ofsuch other diseases using the appropriate levels and ratios for thatdisease.

In one aspect of the invention, the stage and prognosis of a humanpapillomavirus (HPV) infection or HPV-based disease is assessed. Thisembodiment of the present invention involves the measurement of thelevel of expression of one or more HPV genes discovered to be related tothe stage and nature of HPV-based disease. Genes useful for this purposeinclude the HPV E6, E7, L1, and E2 genes, preferably full-length genes.It has been discovered that the level of expression of these genes, theratio of expression of these genes to each other or to one or more othergenes, or both, are indicative of the stage of HPV-based disease. Thelevel of expression is relative to other HPV genes, or the level ofexpression relative to a non-HPV gene, referred to herein as a referencegene. Such reference genes may be any appropriate gene (not encoded byHPV), and are, for example, housekeeping genes or other constitutivelyexpressed genes. Examples of reference genes include actin genes,cytoskeletal genes, histone genes, tubulin genes, epidermal growthfactor receptor genes, the normal p53 gene, the normal pRB gene, cyclingenes, β-globin genes, and glucose-6-phosphate dehydrogenase genes.Expression of reference genes may be measured in the same cell as thelevel of HPV genes are measured or in neighboring cells in the same cellsample. In such a case, the reference gene is an internal control forgene expression.

For example, the level of expression of the HPV gene and the referencegene is measured in the same cell sample. Such measurements provide aninternal control of the overall expression level in a cell sample andare used to calculate a corrected level of expression for the HPV geneto allow more accurate comparisons of the level of expression betweendifferent cell samples. One form of correction is referred to asnormalization. Thus, the level of expression of one or more HPV genesmay be measured in two or more cell samples along with the level ofexpression of the same reference gene in each of the cell samples. Thelevel of expression of the HPV genes is then normalized to each otherbased on differences, if any, between the measured level of expressionof the reference gene in each of the samples.

Using information about the levels and ratios of HPV genes in differentcell states, the stage of the disease may be assessed in several ways.In some cases, the presence or absence of detectable expression isindicative of the disease state in the infected cells. For example, alack of E2 expression (when HPV is present) is indicative of high gradecervical intraepithelial neoplasia or cancer. In other cases, a changeor difference in expression of an HPV gene product may be indicative ofchange occurring in the infected cell state. For example, an increasedlevel of expression of E6 and E7—relative to, for example, an earliersample or a reference sample—may be indicative of high grade cervicalintrapeithelial neoplasia or cancer. A change in the ratio of E6 and E7expression to E2 expression is used to identify low grade cervicalintrapeithelial neoplasia or a shift from normal cells to low gradecervical intrapeithelial neoplasia. Many other combinations ofcomparisons are also possible.

There are several ways in which measured levels of expression of HPVgenes may be compared and categorized. For example, where the expressionis indicative of the cell state, expression of the HPV gene is analyzedwithout reference to the expression level of other genes. Where therelative level of expression of an HPV gene is indicative of the cellstate, the measured level of expression is compared, for example, to thelevel of expression of the same type of HPV gene in a different cellsample (such as an earlier cell sample from the same source or referencecells harboring HPV), to the level of expression of a different type ofHPV gene in the same or a different cell sample, to the level ofexpression of a non-HPV reference gene in the same cell sample, or tothe level of expression of a non-HPV reference gene in reference cells.

In one embodiment of the present invention, levels and ratios ofexpression of HPV genes may be compared to the levels of the same genesin a cell line that contains HPV (such as HeLa or CaSki). Such celllines provide a standard against which levels of expression of HPV genesin cell samples are compared. Such comparisons are used to assess andcompare the absolute levels of expression of these HPV proteins withthose in a standard or comparative cell line. Other cell lines usefulfor this purpose are non-cancerous cell lines infected with HPV 16 (suchas W12) or HPV 31 (such as CIN-612).

The types of comparison described above may also be used with othergenes and other disease states. That is, the measured level ofexpression of a gene of interest may be compared, for example, to thelevel of expression of the same type of gene in a different cell sample(such as an earlier cell sample from the same source or appropriatereference cells), to the level of expression of a different type of genein the same or a different cell sample, to the level of expression of areference gene in the same cell sample, or to the level of expression ofa reference gene in reference cells.

For hybridization detection of HPV nucleic acids, a mixture of probesspecific for these sequences from different HPV types may be used. Thisensures that the method will detect expression regardless of the type ofHPV involved. For some purposes, it may be desirable to use probesdesigned for the sequence of a certain HPV type, or a mixture of probesfor only some HPV types. Such probes may or may not be type-specificdepending on the differences between the sequences of the HPV nucleicacids to be detected. One useful mixture for this purpose would includeprobes for HPV types more closely associated with a progression tocancer. The HPV types most commonly associated with cervical cancer aretypes 16 and 18.

The clinical significance of introducing high-risk HPV DNA testing toroutine cervical diagnosis by the conventional Pap test is wellrecognized. However, neither of these tests provides information aboutpersistence of high-risk HPV infection highly associated withdevelopment of high-grade neoplasia and cancer. Additional moleculartesting to measure aberrant marker expression in high-risk HPVDNA-positive cases would possibly increase the accuracy and prognosticvalue of a diagnosis. The combination of testing for high risk HPV DNAwith protein markers greatly reduces the rate of false positives andimproves the specificity of HPV DNA for more likely high-grade cervicallesions.

In a preferred embodiment of the invention, high-risk HPV DNA ismeasured by the hybrid capture based HC2 HPV DNA test (DigeneCorporation; Gaithersburg, Md.). Briefly, clinical specimens arecombined with a base solution which disrupts the virus or bacteria andthereby releases target DNA. No special specimen preparation isnecessary. The target or sample DNA is then hybridized with RNA probesforming RNA:DNA hybrids. Multiple RNA:DNA hybrids are captured onto asolid phase coated with universal capture antibodies specific forRNA:DNA hybrids. Captured RNA:DNA hybrids are detected with multiplelabeled antibodies, where alkaline phosphatase is one example of alabel. The resulting signal may be amplified to at least 3000-fold. Thebound label is detected with a substrate, such as chemiluminescentdioxetane substrate. Upon reaction, the substrate produces light that ismeasured on a luminometer in Relative Light Units (RLUs), therebyenabling the detection, identification, and interpretation of HPV DNA inthe subject sample. Although this HPV DNA hybridization method ispreferred, one skilled in the art would understand that an RNA sampleand DNA probes may also be used. Furthermore, multiple testing on asingle platform, a microarray for example, is preferred. The presentinvention is applicable to any known or future test that detects HPV bynucleic acid hybridization analysis, DNA or RNA, or that employs anucleic acid detection test, (i.e., RNA) utilizing a ratio of expressionof early versus late regions of the HPV genome.

Another embodiment of the present invention is the secondary screeningdiagnostic test for molecular markers. The molecular markers assayed formay include additional or surrogate marker characteristics that are nota direct cause or result of the disease, but that are related to certaindisease and cell states. Non-limiting examples of such markers includepolymorphic markers, human leukocyte antigens (HLA) such as B7 thatpredispose women for cervical carcinomas, oncogenes, p53 mutations,other cancer markers, oncosupressors, cytokines, growth factorreceptors, and hormones. Such markers may be present in, or absent from,cells exhibiting state- or disease-specific characteristics, and suchpresence or absence may be indicative of, for example, a more severe orless severe disease state. These markers are used in conjunction withthe disclosed method to infer either higher or lower risk of neoplasticdisease depending on the number of abnormal scores or the magnitude ofchange in quantitative markers.

In particular, the molecular markers of the sequential or concurrentlysequential method may preferably comprise of two levels of proteinmarkers: (a) a proliferation group, of which a preferred member is PCNA;however, MIB-1 (also known as Ki-67), cdc6, and mcm proteins, preferablymcm2 and mcm5 may be also used; and (b) a cell cycle control group, ofwhich a preferred member is p16^(INK4A); however, p21^(WAF1), p14^(ARF)may be also used. Expression of the protein markers may be measuredutilizing primary antibodies, highly specific for the target antigen.The expression of protein markers, such as but not limited to, cellproliferation and cell cycle control group markers, may be screened forin a subject sample by, for example, immunostaining,immunocytochemistry, by an enzyme-linked immunosorbent assay (ELISA) oran analogous test such as a LUMINEX-based protein assay, or proteinmicroarray.

Briefly, the immunostaining procedure involves the steps of preparingthe subject sample, blocking, permeabilizing, adding primary antibodiesraised against the proliferation group and/or the cell cycle controlgroup proteins, adding a labeled secondary antibody which recognizes theprimary, and upon substrate reaction, detecting the signal. Thepreferred members of each molecular marker group, PCNA and p16^(INK4A),were selected for their high affinity of the corresponding primaryantibodies for the target antigen. However, additional related markersmay be utilized in the test to increase the sensitivity as necessary.These primary antibodies may be utilized individually (detection of asingle marker) or as a pool (detection of multiple markerssimultaneously).

Proliferation and cell cycle control group proteins are preferredmolecular markers of disease and cell states since abrogation oftumor-suppression functions of p53 and pRB proteins by high-risk HPVoncoproteins, E6 and E7, leads to cell cycle activation at the G1/Sphase followed by promotion of the host cell and viral DNA synthesis.This phenomenon results in over-expression of the proteins associatingor controlling cell proliferation, DNA replication, and the cell cycle.Cell proliferation markers are gene products which are expressed inactively dividing cells, or cells which are committed to or are enteringthe cell cycle. These markers are generally absent from cells which arequiescent, dormant, in stationary phase or otherwise arrested eithertemporarily or permanently and are not participating in the cell cycle.Non-limiting examples of cell proliferation markers include: PCNA, cdc6,mcm2, mcm3, mcm4, mcm5, mcm6, mcm7, Cdc7 protein kinase, Dbf4, Cdc14protein phosphatase, cyclin A, Cdc45, Ki67, KiS1, and mcm10.

Cell cycle control proteins may therefore be used as markers in thescreening diagnostic test of the present invention. These proteinsregulate and control the cell cycle progression at particularcheckpoints, directing the cell towards proliferation, growth arrest orapoptosis. Non-limiting examples of cell cycle control markers include:p16^(INK4A), p14^(ARF), and p21^(WAF1).

A preferred cell proliferation associated protein is proliferating cellnuclear antigen (PCNA). PCNA is an S-phase associated nuclear proteinand a cofactor of DNA polymerase. Overall, PCNA expression isprogressively correlated with a cervical lesion grade, with about70%-92% positive cases identified in high-grade lesions and cancer²².Over-expression of other proteins related to cell proliferation and DNAreplication, such as, but not limited to, MIB-1 (also known as Ki-67),cdc6, mcm2, and mcm5 has been described in cervical neoplasia andcarcinoma 23. For example, Ki-67 is a non-histone protein expressing inall phases of the cell cycle (with exception of the G0 phase). Cdc6,mcm2, and mcm5 are members of the pre-initiation complex of DNAreplication, therefore, involved in the earlier initiation stage of theprocess. The described group of proteins (PCNA, Ki-67, cdc6, mcm2, andmcm5) is a good indicator of the cell proliferation status, however, notnecessarily associating with neoplastic transformation. Thus, this groupof proteins is distinguished as the proliferation group, although theproteins are over-expressed and are not specific for cervical neoplasticgrade. Cell proliferation markers may be detected at the mRNA stage, butpreferably cell proliferation markers are detected as proteins orpolypeptides.

The regulatory machinery of the cell cycle is composed of cyclins,cyclin-dependent kinases (CDKs) and their inhibitors (CKIs). CKIs may beseparated into two groups having the following proteins: p21^(Waf1),p27^(Kip1) and p57^(Kip2) and the other including p15^(Ink4b),p16^(Ink4a), p18^(Ink4c) and p19^(Ink4d). A preferred cell cycle controlassociated protein is p16^(INK4A). p16^(INK4A) is a cyclin-dependentkinase inhibitor. This protein inhibits cell progression through theG1-S stage of the cell cycle and interaction with cdk4/6. p16^(INK4A) isover-expressed in HPV-associated lesions, with 70%-96% positive casesfor high-grade lesions^(21,24). Additionally, over-expression of otherproteins related to cell cycle control, such as p14^(ARF) andp21^(WAF1), has been found in high grade squamous intraepithelial lesion(suspect CIN 2-3) and cervical carcinoma. Both of these proteins areinvolved in cell cycle arrest in the G1 and G2/M phases through MDM2(p14^(ARF)) or cyclins and cdk complexes (p21^(WAF1))²⁵. The previouslydescribed group of proteins (p16, p14, and p21) relates to cell cyclecontrol and associates with neoplastic transformation. Thus, this groupof proteins is referred to as the cell cycle control group, specificallyover-expressed in HPV-associated cervical neoplasia and cancer.

In yet another embodiment of the present invention, the sequential orconcurrently sequential method is advantageous in that the number offalse positive results is significantly reduced. Performing the HPVnucleic acid test and performing the protein marker test for cellproliferation and/or cell cycle control group proteins individuallyresults in false positive results. However, the combination of thesetests as described herein, increases the specificity and reduces thecross reactivity associated with false positive results. For example,Table 4 shows positive cases identified by HPV DNA and preferredmolecular markers. Specifically, 8% of the normal clinical specimens(WNL) were positively identified by high risk HPV DNA screening alone;26% of the WNL specimens were positively identified by PCNAproliferation group protein marker screening alone; 11% of the WNLspecimens were positively identified by p16 cell cycle control groupprotein marker screening alone; 27% of the WNL specimens were positivelyidentified by PCNA and/or p16 protein marker screening; and 3% of theWNL specimens were positively identified by HPV DNA screening and PCNAand/or p16 protein marker screening. The number of positive specimens issignificantly reduced when the screening tests are combined. This may beattributed to the specificity incurred by the combination of tests. Thenumber of false positives is reduced by the range of about 15%-100%,about 20%-90%, or about 30%-70%. One advantage of this sequential orconcurrently sequential method is that the individual tests may be lessstringent or restrictive since the combination of tests increases thespecificity.

A further embodiment of the present invention relates to a method ofassessing risk associated with cellular abnormality in a subject'ssample comprising obtaining sample cells from a subject, detectinghigher or lower risk HPV infection; and identifying the over-expressedmolecular markers, such as proliferation and/or cell cycle control groupproteins. In particular, HPV nucleic acid data are combined with proteinmarker expression data to provide an improved and more accurateassessment of risk for concurrent or future cervical neoplasia withfewer false results. The positive or negative results of the moleculartests, such as high-risk HPV nucleic acid and protein marker expressiontests, may be based on the signal to cutoff value.

In general, a clinical specimen that is tested is positive if itsspecimen signal intensity to cutoff value ratio is greater than or equalto 1. The cutoff value is determined experimentally for any type ofassay and may be varied from assay to assay. The cutoff value isdetermined using detection of assay positive controls. An assay positivecontrol is run in replicates and its mean value should be above the meanvalue from detection of negative control. The difference betweenpositive control and negative control values should be statisticallyreliable. Statistical reliability depends on the number of replicates inthe assay and the cutoff value percent (% CV) of each value.

In one embodiment of the present invention that uses the HC2 HPV DNAassay, the cutoff value is experimentally determined using an HPVpositive control having a concentration of 1 pg/ml. The assay detectionresults are expressed as a ratio of the specimen signal intensity inrelative light units (RLU) to its cutoff value. Specimens with aspecimen signal/cutoff value ratio of greater than or equal to 1 is HPVpositive, and indicative of HPV infection, risk of neoplasia, and thepresence or expression of HPV nucleic acids. Specimens with a specimensignal/cutoff value ratio of less than 1 is HPV negative (DigeneCorporation; HR HPV DNA Test).

In particular for the protein immunostaining assay, the cutoff value isexperimentally determined in the 99^(th) percentile optical density (OD)values generated by measuring a protein marker negative control. Theassay result is expressed as a ratio of the specimen signal intensity(i.e., median OD value above the cutoff value) to the cutoff value. Thissignal to cutoff value ratio is also referred to as the median ratio.Specimens with a specimen signal intensity to cutoff value ratio ofgreater than or equal to 1, preferably greater than or equal to 1.2, areprotein marker positive as described in Example 2. Specimens with aspecimen signal intensity to cutoff value ratio of less than 1 areprotein marker negative.

In brief, there are two methods of analyzing immunostaining: visuallyand by densitometry as described above. According to the visual method,the blue negative and red-brown positive stained cells are manuallycounted. The staining intensity is graded and scored on a scale of ½+,1+, 2+, and 3+, where 3+ is very strong. Specimens having a score of 3%at 1+ or higher are considered to be positive for PCNA+MIB-1. Thosespecimens with a positive score of 1+ are considered to be positive forp16.

In yet a further embodiment of the present invention, the results of theprimary and secondary screenings are assessed. More particularly, themolecular screening tests for cervical cancer described herein enablefurther characterization of the specimens in relation to neoplasticgrade. Molecular grades may be useful for more accurate medicalfollow-up procedures. The sequential or concurrently sequential methodof detecting HPV nucleic acids and molecular protein marker(s) is basedon whether a subject sample is positive or negative for (1) the primaryHPV nucleic acid screen; and/or (2) the secondary screen for proteinmarker for proliferation group and/or cell cycle control group proteins.

A molecular grade 0 (MG0) is considered to be a subject sample that isnegative for the primary high risk HPV nucleic acid test, and will notbe tested for the secondary protein markers test. A molecular grade I(MGI) is considered to be a subject sample that is positive for theprimary HPV nucleic acid screen, but negative for the secondary proteinmarker screen. A molecular grade II (MGII) is considered to be a subjectsample that is positive for both the primary HPV nucleic acid andsecondary protein marker screens. The recommendation of cervicalscreening management using the novel sequential or concurrentlysequential diagnostic screening method and molecular classification ofthe present invention is to repeat routine screening for high risk HPVnucleic acid test in 3-5 years from the initial testing if the subjectsample is a MG0, which indicates the absence of high-risk HPV infectionand cervical neoplastic lesions. For individuals having MGI, thesequential or concurrently sequential screening method should berepeated in 3-12 months from the initial screen. This grade indicatesthe presence of high risk HPV DNA, but not necessarily a neoplasticlesion and it is important to determine if the HPV infection ispersistent as indicated by a repeat positive HPV DNA. For individualshaving a MGII, immediate referral for a colposcopy and under thephysician's recommendation, any subsequent biopsy and treatment. MGIIindicates the presence of high-risk HPV DNA, elevated expression ofprotein markers, and increased risk of neoplastic lesions and/orcervical cancer.

In yet another embodiment of the present invention, automated screeningdevices are preferably used in conjunction with the method ofidentifying HPV-related molecular markers.

The contents of all patents, patent applications, published PCTapplications and articles, books, references, reference manuals andabstracts cited herein are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

As various changes may be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Many modifications and variationsof the present invention are possible in light of the above teachings.

EXAMPLES Example 1 Protocol for Immunocytochemical Staining

Slide Preparation

Teflon-printed 8-well slides (EMS; Fort Washington, Pa.) were incubatedin Histogrip (Zymed Laboratories, Inc.; South San Francisco, Calif.) for2 min at room temperature (RT). The slides were rinsed in water (threechanges) and air dried at RT.

Specimen Preparation

Aliquots of liquid cervical specimens collected either in PRESERVCYT(PC), (Cytyc Corporation; Boxborough, Mass.) or Universal CollectionMedia (UCM: Digene Corporation; Gaithersburg, Md.) were centrifuged at5223 rcf for 5 minutes to obtain cell pellets (approximately100,000-200,000 cells). The supernatants were removed and the cellpellets were resuspended in 1 ml of MUCOLEXX (Shandon Inc.; Pittsburgh,Pa.). The specimens were incubated in MUCOLEXX for approximately 15-18hours at RT. The MUCOLEXX was removed by centrifugation at 5223 rcf for5 minutes. The specimens were further processed through a modifiedprotocol (TriPath; Burlington, N.C.). Briefly, the cell pellets wereresuspended in 750 μl of CytoRich Preservative Fluid (TriPath) and addedto the top of 750 microliters of CYTORICH Density Reagent (TriPath). Thegradient mixture was centrifuged at 200 rcf for 2 minutes, followed byremoval of 650 microliters of the mixture from the top. The remainder ofeach specimen was centrifuged at 800 rcf for 10 minutes, followed byremoval of supernatants. The pelleted specimens were washed once andthen resuspended in PC (Cytyc Corporation). The resuspended specimenswere applied to the wells of an 8-well slide (approximately 500-1,000cells per well), and air-dried at RT for 10-30 minutes. Dried slideswere fixed in 95% ethanol for at least 10 minutes prior toimmunostaining.

Immunostaining Protocol

ENVISION+ SYSTEM, HRP kit (DAKO Corporation; Carpinteria, Calif.) andprotocol were utilized for immunostaining. Briefly, the slide was airdried, then dipped in Tris Buffered Saline (TBS), (DAKO Corporation).Excess TBS was gently blotted and 250 microliters of 4 mM SodiumDeoxycholate (Sigma; St. Louis, Mo.) was spread evenly over the wells.The specimens were incubated with Sodium Deoxycholate for 5 minutes atRT, followed by dipping the slide in 0.25% Triton X in TBS for 30seconds. The slide was gently blotted and incubated with PeroxidaseBlocking Agent (DAKO Corporation) for 5 minutes at RT, followed bydipping and washing in TBS for 30 seconds. Excess TBS was gentlyblotted, and 250 microliters of primary antibody properly diluted inAntibody Diluent was applied to the wells. The following primaryantibodies were utilized: monoclonal mouse anti-human Ki-67 (MIB-1, code# M 7240; DAKO Corporation); monoclonal mouse anti-human PCNA (PC10,code # MS-106-P; Lab Vision Corporation; Fremont, Calif.), monoclonalmouse anti-human p16^(ink4a) (E6H4, code #OA 315; DAKO A/S, Denmark).Some specimens were tested using a different clone of p16^(ink4a):DCS-50.1/H4 from Oncogene Research Products; Boston, Mass. The slide wasincubated at RT for 30 min (antibody clone DCS-50.1/H4 was used at 37°C.) in a humidity chamber, followed by 3 changes of washes in TBS for 2minutes each. Excess TBS was gently blotted and secondary anti-mouseHRP-labeled antibody was applied per slide. The slide was incubated atRT for 30 minutes in a humidity chamber, followed by 3 changes of washesin TBS for 2 minutes each. Excess TBS was gently blotted and 250microliters of Liquid DAB+ substrate-chromogen solution (DAKOCorporation) was applied to the slide, which was then incubated for 7minutes at RT. The slide was dipped and washed in TBS for 1 minute,followed by washing in 95% ethanol. The slide was counterstained inHematoxylin for 35 seconds, and washed in two changes of water. Finally,the slide was stained in 37 mM Ammonium Hydroxide (EMS; Fort Washington,Pa.), followed by washing in water and air-drying the excess liquid. Thedried slide was mounted with Permanent Mounting Media (DAKO Corporation)and covered with a microglass cover slip (VWR Scientific Products;Willard, Ohio). The immunostaining was evaluated under the microscope.Cells expressing protein markers were positively stained with browncolor.

Example 2 Procedure for Scoring Results

Immunostaining Evaluation by Visual Score

The immunostained specimens were scored visually. Briefly, negatively(blue) and positively (red-brown) stained cells were counted. Eachspecimen counting score contained at least 100-200 cells. The percentageof positive cells within the negative cell population was calculated.The staining intensity was graded at ½+ (light), 1+ (mild), 2+(moderate), and 3+ (strong). Specimens with a score of 3% at 1+ orhigher were considered to be positive for PCNA+MIB-1. Specimens with apositive score of 1+ were considered to be positive for p16.

Immunostaining Evaluation by Densitometry Software

The immunostained specimens were scored utilizing Image-Pro® Plussoftware (OPELCO; Dulles, Va.). Briefly, the light intensity of themanually selected cells from the color (Red, Green, Blue) photographicimage was measured for cervical controls and specimens. The intensity ofblue color (Hematoxylin counterstain) was filtered out by the software,and the average intensity of red and green (DAB positive stain) colorswas calculated. The relative optical density (OD) was extrapolated fromthe standard calibration curve. An OD value of the assay cutoff wasestablished by calculating the 99^(th) percentile OD for the negativecontrol. A median value of the specimen OD above the cutoff wascalculated. The median ratio was calculated by dividing the specimen'smedian OD value over the cutoff. Specimens with a median ratio greaterthan or equal to 1, preferably greater than or equal to 1.2, werepositive for protein expression.

Example 3 Identification of Positive Cases by HPV DNA and Protein MarkerAnalyses

Results

PRESERVCYT® (Cytyc Corporation) clinical cervical specimens wereutilized for assessment of high risk HPV DNA and protein markersexpression. The specimen's cytological diagnosis was confirmed bytrained professionals using the Pap test results. The specimens weregrouped based on cytological diagnosis rendered by trained expertcytologists as follows: a) within normal limits (WNL), b) LSIL (suspectcervical intraepithelial neoplasia 1; CIN1), comprised of milddysplasia, c) HSIL (suspect CIN 2), comprised of moderate dysplasia, andd) HSIL (suspect CIN 3+), comprised of moderate to severe and severedysplasia, in situ and invasive carcinoma. The cytological diagnosis wasused as the “gold standard” for evaluation of the novel molecularmethod. The immunostaining results scored visually are presented inTable 1. TABLE 1 Positive Cases Identified by HPV DNA and ProteinMarkers PCNA + MIB-1 PCNA + MIB-1 p16 Cell PCNA + MIB-1 and/or p16 andTotal # High Risk Proliferation Cycle Control and/or High Risk HPV ofHPV DNA, # (%) Group, # (%) Group,# (%) p16, # (%) DNA, # (%) ClinicalSpecimen Specimens Positive Positive Positive Positive Positive WNL 46 3(6.5%) 12 (26.1%) 5 (10.9%) 14 (30.4%) 0 (0%) HSIL (suspect CIN 2) 62 55(88.7%) 43 (69.4%) 33 (53.2%) 48 (77.4%) 43 (69.4%) HSIL (suspect CIN2-3+) 81 74 (91.4%) 61 (75.3%) 49 (60.5%) 67 (82.7%) 62 (76.5%) HSIL(suspect CIN 3+) 19 19 (100%) 18 (94.7%) 16 (84.2%) 19 (100%) 19 (100%)

A total of 46 cervical specimens with WNL cytological diagnoses weretested for the presence of high-risk HPV DNA using the HC2 test. Thepresence of high-risk HPV DNA was found in 3 out of 46 specimens (6.5%).The same group of specimens (total number 46) was tested for expressionof the proliferation group of protein markers (PCNA+MIB-1) and the cellcycle control group of markers (p16^(INK4A)) using the immunostainingprotocol. Positive expression of proliferation markers was found in 12out of 46 specimens (26.1%). Positive expression of p16^(INK4A) wasfound in 5 out of 46 specimens (10.9%). Overall, 14 out of 46 WNLspecimens (30.4%) were found positive for a combination of either groupof protein markers (proliferation or cell cycle control), while usinghigh-risk HPV DNA detected only 6.5% HPV positive samples. None of 14specimens, positive for either molecular marker, was positive forhigh-risk HPV DNA. Thus, 0 out of 46 WNL specimens (0%) were foundpositive for both high-risk HPV DNA and any one or more of the proteinmarkers. The rest of the results presented in Table 1 were interpretedin a similar fashion. Briefly, 88.7% of the HSIL (suspect CIN 2) groupwas found positive for high-risk HPV DNA, and 69.4% of the same groupwas positive for a combination of high-risk HPV DNA and eitherproliferation or cell cycle control markers. Additionally, 91.4% of theHSIL (suspect CIN 2-3+) group was found positive for high-risk HPV DNA,and 76.5% of the same group was positive for a combination of high-riskHPV DNA and either proliferation or cell cycle control markers. Finally,100% of the HSIL (suspect CIN 3+) group was found positive for high-riskHPV DNA, and 100% of the same group was positive for a combination ofhigh-risk HPV DNA and either proliferation or cell cycle controlmarkers.

A new set of PC cervical specimens (including six specimens from the WNLgroup that were scored visually in Table 1) was tested for high-risk HPVDNA by the HYBRID CAPTURE (HC2; Digene Corporation) test and forexpression of protein markers by the immunostaining test. The specimenswere grouped based on the received cytological diagnosis as followed: a)WNL, b) LSIL (suspect CIN 1), and c) HSIL (CIN 2-3). The immunostainedslides were analyzed by the densitometry software and scoring algorithm(Image-Pro® Plus; OPELCO; Dulles, Va.). The compatibility of thesoftware and algorithm with the visual method for immunostaininganalysis is shown in Table 2. TABLE 2 Positive Cases Identified by HPVDNA and Protein Markers PCNA +MIB-1 PCNA + MIB-1 p16 Cell PCNA +MIB-1&/or p16 & Total # High Risk Proliferation Cycle Control &/or High RiskHPV of HPV DNA, # (%) Group, # (%) Group, # (%) p16, # (%) DNA, # (%)Clinical Specimen Specimens Positive Positive Positive Positive PositiveWNL 23 3 (13%) 0 (0%) 1 (4.3%) 1 (4.3%) 0 (0%) LSIL (suspect CIN 1) 2119 (90.5%) 11 (52.4%) 2 (9.5%) 11 (52.4%) 9 (42.9%) HSIL (suspect CIN2-3) 20 17 (85%) 16 (80%) 15 (75%) 19 (95%) 16 (80%)

The results of Table 2 were interpreted in the same fashion as forTable 1. Briefly, 13% of the WNL specimens were found positive forhigh-risk HPV DNA, and 0% of the same specimens were positive for acombination of high-risk HPV DNA and either proliferation or cell cyclecontrol markers. Additionally, 90.5% of the LSIL (suspect CIN 1)specimens were found positive for high-risk HPV DNA, and 42.9% of thesame specimens were positive for a combination of high-risk HPV DNA andeither proliferation or cell cycle control markers. Finally, 85% of theHSIL (suspect CIN 2-3) specimens were found positive for high-risk HPVDNA, and 80% of the same specimens were positive for a combination ofhigh-risk HPV DNA and either proliferation or cell cycle controlmarkers.

Alternatively, testing for protein markers may be performed using PCNA,MIB-1, and p16 as a single pool of antibodies with similar sensitivityas shown with two independent groups or double pools of antibodies. Thesame set of cervical specimens, analyzed using the software and scoringalgorithm (Table 2), was used for antibody pool comparison. Thefollowing groups of specimens were tested: a) WNL, b) LSIL (suspect CIN1), and c) HSIL (suspect CIN 2-3). The comparison data for double pools(PCNA+MIB-1 or p16) and a single pool (PCNA+MIB-1+ p16) of antibodiesare presented in Table 3. TABLE 3 Protein Expression Data for SingleMarker Pool Clinical Double Pools + Single Pool + Specimens Total # HighRisk HPV High Risk HPV (same as of DNA, # (%) DNA, # (%) in Table 2)Specimens Positive Positive WNL 5 0 (0%) 0 (0%) LSIL 21 9 (42.9%) 8(38.1%) (suspect CIN 1) HSIL 7 6 (85.7%) 6 (85.7%) (suspect CIN 2-3)

The high-risk HPV DNA and immunostaining results for double pools,either proliferation or cell cycle control group of markers, werepreviously identified (Table 2). A new set of data was generated byusing a combination of high-risk HPV DNA and immunostaining results fromthe single pool of primary antibodies (PCNA, MIB-1, and p 16). Verysimilar results were scored for a combination of high-risk HPV DNA andeither the single pool or double pools of antibodies. Briefly, thecomparison data scored by either combination of antibodies pools withhigh-risk HPV DNA were identical for WNL (0% positive) and HSIL (suspectCIN 2-3) (85.7% positive) groups. Very marginal variation in percentpositive cases between single pool plus HPV (38.1%) and double poolsplus HPV (42.9%) was noted for LSIL (suspect CIN 1) group.

The immunostaining assay may also be performed using the preferredprotein markers, PCNA and p16, individually. A new set of PC cervicalspecimens was used for this example. The specimens were grouped based onthe received cytological diagnosis as followed: a) WNL, b) ASCUS, c)LSIL (suspect CIN 1), and d) HSIL (suspect CIN 2-3). All groups weretested for high-risk HPV DNA by the HC2 test. PCNA and p16 proteinexpression was performed by the immunostaining assay. The results scoredby the software algorithm (Image-Pro® Plus (OPELCO; Dulles, Va.)) arepresented in Table 4. TABLE 4 Positive Cases Identified by HPV DNA andPreferred Protein Markers PCNA &/or PCNA P16 cell p16 & High Total #High risk proliferation cycle control PCNA &/or Risk HPV of HPV DNA, #(%) group, # (%) group, # (%) p16, # (%) DNA, # (%) Clinical specimensspecimens positive positive positive positive positive WNL 102 8 (8%) 26(26%) 11 (11%) 27 (27%) 3 (3%) ASCUS 88 42 (48%) 39 (44%) 22 (25%) 46(52%) 25 (28%) LSIL (suspect CIN 1) 92 79 (86%) 47 (51%) 15 (16%) 49(53%) 44 (48%) HSIL (suspect CIN 2-3) 85 81 (95%) 69 (81%) 56 (66%) 75(88%) 71 (84%)

A total of 102 WNL cervical specimens were tested for high-risk HPV DNA.The presence of high-risk HPV DNA was found in 8 out of 102 (8%) WNLspecimens. The same group of specimens was tested for expression ofpreferred protein markers (PCNA and p 16) by the immunostainingprotocol. Positive PCNA expression was found in 26 out of 102 (26%) WNLspecimens. Positive p16 expression was found in 11 out of 102 (11%) WNLspecimens. Overall, we found 27 out of 102 (27%) WNL specimens positivefor PCNA or p16 expression. The combination of high-risk HPV DNA andpreferred protein markers tests demonstrated 3 out of 102 (3%) positivefor the WNL group. The rest of the results were interpreted in a similarfashion. Briefly, 48% of ASCUS specimens were found positive forhigh-risk HPV DNA, and 28% of the same specimens were positive for acombination of high-risk HPV DNA and either proliferation or cell cyclecontrol markers. Additionally, 86% of the LSIL (suspect CIN 1) specimenswere found positive for high-risk HPV DNA, and 48% of the same specimenswere positive for a combination of high-risk HPV DNA and either marker.Finally, 95% of the HSIL (suspect CIN 2-3) specimens were found positivefor high-risk HPV DNA, and 84% of the same specimens were positive for acombination of high-risk HPV DNA and either marker.

For example, Table 4 illustrates the increasing specificity andreduction of clinical false positive results as the HPV nucleic acidtest is combined with the molecular marker test. Of the WNL specimens,8% are positive as determined by the HPV nucleic acid test alone, whileprotein marker tests of the WNL specimens result in 11%-27% positiveresults. However, the combination of the high risk HPV DNA primaryscreen and the protein marker secondary screen results in only 3% of theWNL specimens as positive. This significant reduction in positiveresults is due to the reduction of clinical false positive results or inother words, specimens that are HPV DNA positive but are from women whodo not appear to have clinical disease. The HPV DNA test results are notsuggested to be analytically false positive, but in the vast majority ofcases, the HPV DNA is actually present in such HPV DNA test positivecases in clinically non-diseased women. Accordingly, this sequential orconcurrently sequential method is accurate, sensitive, and reduces thenumber of false positives by the range of about 15%-100%, about 20%-90%,or about 30%-70%.

Additional markers, such as mcm2, mcm5, and p14ARF, can be utilized incombination with either high-risk HPV DNA and PCNA or high-risk HPV DNAand any marker (PCNA, p16, mcm2, mcm5, p14ARF). The same set of cervicalspecimens, as shown in Table 3, was used for evaluation of theadditional markers. mcm2 expression was measured using monoclonal mouseanti-human mcm2 antibody (code # ab6153, Abcam Limited, Cambridge, UK).mcm5 expression was measured using monoclonal mouse anti-human mcm5antibody (code # ab6154, Abcam Limited, Cambridge, UK). Finally, p14ARFexpression was measured using monoclonal mouse anti-human p14ARFantibody (14PO3, code # MS-1115-P, Lab Vision Corporation, Fremont,Calif., USA). The clinical evaluation of mcm2, mcm5, and p14ARF is shownin Table 5. TABLE 5 Positive Cases Identified by HPV DNA, PCNA, p16,mcm2, mcm5, and p14 Mcm2 &/or Mcm5 &/or P14 &/or PCNA & Any Marker* &Total # High Risk PCNA & High Risk PCNA & High Risk High Risk HPV HighRisk HPV of HPV DNA, # (%) HPV DNA, # (%) HPV DNA, # (%) DNA, # (%) DNA,# (%) Clinical Specimens Specimens Positive Positive Positive PositivePositive WNL 38 6 (16%) 3 (8%) 4 (11%) 3 (8%) 4 (11%) ASCUS 40 22 (55%)14 (35%) 15 (38%) 15 (38%) 15 (38%) LSIL (suspect CIN 1) 37 31 (84%) 21(57%) 24 (65%) 22 (59%) 24 (65%) HSIL (suspect CIN 2-3) 40 38 (95%) 32(80%) 33 (83%) 34 (85%) 36 (90%)*PCNA, p16, mcm2, mcm5, p14

A total of 38 WNL cervical specimens were tested for high-risk HPV DNA.The presence of high-risk HPV DNA was found in 6 out of 38 (16%) WNLspecimens. The same group of specimens was tested for expression ofpreferred protein markers (PCNA and p16) and additional protein markers(mcm2, mcm5, and p14) by the immunostaining protocol. The combination ofhigh-risk HPV DNA and either PCNA and/or mcm2 tests demonstrated 3 outof 38 (8%) positive for WNL specimens. The combination of high-risk HPVDNA and either PCNA and/or mcm5 tests demonstrated 4 out of 38 (11%)positive for the same specimens. The combination of high-risk HPV DNAand either PCNA and/or p14 tests demonstrated 3 out of 38 (8%) positivefor this group. Finally, we found 4 out of 38 (11%) WNL specimenspositive for high-risk HPV DNA and combination of any protein markers,PCNA, p16, mcm2, mcm5, and p14. The rest of the results were interpretedin a similar fashion.

Future molecular staging and classification for potentially progressivecervical disease may be performed as demonstrated in Tables 7 and 8. Thehypothesis is related to grouping the specimens based on theirsimilarities in being positive for the spectrum of molecular events orhits. For example, the cases negative for high-risk HPV DNA could bestaged as MG0. This grade signifies the absence of high-risk HPVinfection and neoplastic lesions. The cases positive for high-risk HPVDNA and negative for either protein marker could be staged as MolecularGrade I (MG). This grade possibly could signify a small low grade lesionwith low exfoliation of abnormal cells or latent high-risk HPV infection(since no additional molecular marker positives are detectable), whicheither could lead to spontaneous viral clearance or persistence. Thecases positive for both high-risk HPV DNA and either group of markerscould be staged as MG II. (Using a panel of markers would possibly allowfor identification of sub-grades based on variation of positive markersper case.) This grade represents specimens with cervical neoplasticlesions, either low- and/or high-grade lesions, since the highestmolecular activity score is measured. It is the dramatically increasedrisk of high-grade neoplastic HSIL (suspect CIN 3+) disease in MGII thatis of greatest importance and interest for users of the disease gradeassessment strategy described herein. TABLE 6 Potential MolecularStaging (MG) of Cervical Neoplasia Total # MG0, # MG I, # MG II, # of(%) (%) (%) Clinical Specimens Specimens Positive Positive Positive WNL102 84%-97% 3%-8% 0%-8% ASCUS 88 45%-58% 19%-20% 23%-35% LSIL (suspectCIN 1) 92 13%-16% 30%-44% 44%-54% HSIL (suspect CIN 2-3) 85 4%-5%11%-13% 82%-84% HSIL (suspect CIN 3+) 19 0% 0% 100%

Table 6 utilized the group of specimens, WNL, ASCUS, LSIL (suspect CIN1), HSIL (suspect CIN 2-3), from Table 5 and the HSIL (suspect CIN 3+)specimens were the same as those used in Table 2. MG0, or neoplasticdisease free women, were found to comprise 84%-97% of the WNL group. Thesame (WNL) group was staged within 3%-8% for MG I and 0%-8% for MG II.MG0 was staged for 45%-58% of woment with ASCUS diagnosis. The ASCUSgroup was staged within 19%-20% and 23%-35% for MG I and MG II,respectively. MG0 was staged for 13%-16% of women with LSIL (suspectCIN 1) diagnosis. The LSIL (suspect CIN 1) group was shown to have aboutequal representation of MG I and MG II (30%-40% and 44%-54%,respectively). In contrast, the majority of HSIL (suspect CIN 2-3 andHSIL (suspect CIN 3+) groups were classified as MG II (82%-84% and 100%,respectively). MG0 was staged in 4%-5% and 0% of women with HSIL(suspect CIN 2-3) and HSIL (suspect CIN3+) diagnoses, respectively. Themolecular screening tests for cervical cancer may allow forcategorization of the specimens in relation to neoplastic grade, asshown above. Thus, molecular grades may be used for more accuratemedical follow-up procedures.

A 95% confidence interval gives an estimated range of values which arelikely to include with 95% confidence, the estimated true but unknownpopulation mean parameter, the estimated range being calculated from agiven set of sample data. The 95% and 99% confidence intervals whichhave 0.95 and 0.99 probabilities of containing the parameterrespectively are most commonly used. If independent sets of samples aretaken repeatedly from the same population, and a confidence intervalcalculated for each set of samples, then 95% of such estimated intervals(confidence level) will contain the true but unknown populationparameter. The confidence interval represents the range of values,consistent with the data, that is believed to encompass the “true” valuewith 95% probability. The confidence interval is expressed in the sameunits as the estimate. Wider intervals indicate lower precision; narrowintervals, greater precision. TABLE 7 Potential Molecular Staging (MG)Of Cervical Neoplasia With 95% Confidence Intervals (CI) Total # MG 0, #MG I, # MG II, # of of of of Clinical Specimens Specimens Specimens MG0, 95% CI Specimens MG I, 95% CI Specimens MG II, 95% CI WNL 102 940.9216 (0.8513, 5 0.0490 (0.0161, 3 0.0294 (0.0061, 0.9655) 0.1107)0.0836) ASCUS 88 46 0.5227 (0.4135, 17 0.1932 (0.1168, 25 0.2841(0.1930, 0.6304) 0.2912) 0.3902) LSIL (suspect CIN 1) 92 13 0.1413(0.0774, 35 0.3804 (0.2812, 44 0.4783 (0.3730, 0.2295) 0.4876) 0.5850)HSIL (suspect CIN 2-3) 85 4 0.0471 (0.0130, 10 0.1176 (0.0579, 71 0.8353(0.7391, 0.1161) 0.2057) 0.9069) HSIL (suspect CIN 3+) 19 0 0.0000(0.0000, 0 0.0000 (0.0000, 19 1.0000 (0.8235, 0.1765) 0.1765) 1.0000)

The 95% confidence interval analysis of the data of Table 7 suggestedthat 92.16% of the WNL specimens were MG0 in the WNL population, wherethe WNL population characterized as MG0 falls within 85.13% to 96.55%;4.9% of the WNL specimens were MGI in the WNL population, where the WNLpopulation characterized as MGI falls within 1.6% to 11.07%; 2.94% ofthe WNL specimens were MGII in the WNL population, where the WNLpopulation characterized as MGII falls within 0.61% to 8.36%; 52.27% ofthe ASCUS speciments were MG0 in the ASCUS population, where the ASCUSpopulation characterized as MG0 falls within 41.35% to 63.04%; 19.32% ofthe ASCUS specimens were MGI in the WNL population, where the ASCUSpopulation characterized as MGI falls within 11.68% to 29.12%; 28.41% ofthe ASCUS specimens were MGII in the ASCUS population, where the ASCUSpopulation characterized as MGII falls within 19.30% to 39.02%; 14.13%of the LSIL (suspect CIN 1) specimens were MG0 in the LSIL (suspectCIN 1) population, where the LSIL (suspect CIN 1) populationcharacterized as MG0 falls within 7.74% to 22.95%; 38.04% of the LSIL(suspect CIN 1) specimens were MGI in the LSIL (suspect CIN 1)population, where the LSIL (suspect CIN 1) population characterized asMGI falls within 28.12% to 48.76%; 47.83% of the LSIL (suspect CIN 1)specimens were MGII in the LSIL (suspect CIN 1) population, where theLSIL (suspect CIN 1) population characterized as MGII falls within37.30% to 58.50%; 4.71% of the HSIL (suspect CIN 2-3) specimens were MG0in the HSIL (suspect CIN 2-3)population, where the HSIL (suspect CIN2-3) population characterized as MG0 falls within 1.3% to 11.61%; 11.76%of the HSIL (suspect CIN 2-3) specimens were MGI in the HSIL (suspectCIN 2-3) population, where the HSIL (suspect CIN 2-3) populationcharacterized as MGI falls within 5.79% to 20.57%; 83.53% of the HSIL(suspect CIN 2-3) specimens were MGII in the HSIL (suspect CIN 2-3)population, where the HSIL (suspect CIN 2-3) population characterized asMGII falls within 73.91% to 90.69%; 0.0% of the HSIL (suspect CIN 3+)specimens were MG0 in the HSIL (suspect CIN 3+) population, where theHSIL (suspect CIN 3+) population characterized as MG0 falls within 0% to17.65%; 0% of the HSIL (suspect CIN 3+) specimens were MGI in the HSIL(suspect CIN 3+) population, where the HSIL (suspect CIN 3+) populationcharacterized as MGI falls within 0% to 17.65%; and 100% of the HSIL(suspect CIN 3+) specimens were MGII in the HSIL (suspect CIN 3+)population, where the HSIL (suspect CIN 3+) population characterized asMGII falls within 82.35% to 100%.

CONCLUSION

By comparing testing results for high risk HPV DNA coupled with cytologywith results obtained for high risk HPV DNA and markers, the incidenceof positive cases was reduced from 8% to 3% for WNL, from 48% to 28% forASCUS, and from 86% to 48% for LSIL (suspect CIN 1) (Table 4). A few WNLspecimens were positive for high-risk HPV DNA (6.5% from Table 1 and 8%from Table 4). These specimens, although without detectable cytologicalabnormalities, may be at higher risk of developing neoplastic lesions.However, 0% to 3% of these HPV positive WNL specimens were positive foreither PCNA and/or p16 protein markers. Thus, a very low rate of HPV DNAin combination with markers that are positive was demonstrated incytologically normal women. In contrast, a relatively high percent ofWNL specimens were positive for either protein markers (30.4% in Table 1and 27% in Table 4), suggesting the limited specificity of the proteinmarkers as an individual test. However, the combination of tests forhigh-risk HPV DNA and protein markers demonstrated very high sensitivity(Table 1) for HSIL (suspect CIN 3+) group, which comprises lesions muchless likely to show spontaneous regression to normal cytology and anincreased risk of progression to cancer. Relatively high sensitivity ofthe same test combination was demonstrated for the HSIL (suspect CIN2-3) group (84% in Table 4).

The data from Table 4 indicate that the addition of a molecular markertest to HPV DNA may significantly improve specificity of HPV DNA forpotential high grade lesions and cancer. The presence of high-risk HPVDNA was demonstrated in 48% and 86% of ASCUS and CIN 1 specimens,respectively. Additional test for protein markers reduces HPV positivesby 20% and 38% for ASCUS and LSIL (suspect CIN 1) groups, respectively.High-risk HPV DNA test helped to reduce the cases that are less likelyto have or progress to high grade lesion or cancer in ASCUS and LSIL(suspect CIN 1) groups by 52% (considering 88 ASCUS women as 100%) andby 14% (considering 92 LSIL women as 100%), respectively. Additionaltests for protein markers reduced HPV positives with concurrent markerstaining by 20% and 38% for ASCUS and LSIL (suspect CIN1) groups,respectively. Furthermore, the combination of high risk HPV DNA withmolecular markers tests helped to reduce the cases that are less likelyto have or progress to high grade lesion or cancer in ASCUS and LSIL(suspect CIN 1) groups by 72% and 52%, respectively. Overall, reductionof test positivity in women who are less likely to have high-gradelesion or cancer is beneficial and can be achieved by implementing thecombination of high-risk HPV DNA with protein markers for the ASCUS andLSIL (suspect CIN1) groups, respectively. Therefore, the datademonstrate the ability of categorizing high-risk HPV DNA positivespecimens into lesions that are more likely to be high grade as opposedto low grade by adding protein markers testing to HPV DNA screening.These data also indicate the potential clinical significance of thenovel molecular diagnostic method for possibly more accurate andspecific diagnosis of ASCUS and LSIL (suspect CIN 1) lesions. Thepotential improved specificity of high-risk HPV DNA through molecularmarker testing enables novel cervical cancer screening to be a usefultool for women of all ages.

Furthermore, the hypothesis and preliminary data supporting thepossibility of molecular classification of potentially progressivecervical disease were described. These results indicate the possibilityof classifying exfoliated cervical cell specimens according to themolecular stage of the underlying SIL lesions when using the combinationof high risk HPV DNA and the markers (Tables 7 and 8) in the absence ofinformation on the cytology grading and thus this strategy constitutes anovel molecular HPV-markers test superior to Pap and HPV-Pap tests. Theinformation on high-risk HPV DNA types may help to detail moleculargrades into sub-grades. Overall, the data support the utility of thenovel molecular method for cervical cancer diagnosis.

Publications cited herein and the material for which they are cited arespecifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCES

-   1. Ponten et al. Int J Cancer 60:1-26, 1995.-   2. Russell L. Educated Guesses: Making Policy About Medical    Screening Tests. Berkeley, University of California Press, 6-24,    1994.-   3. American Cancer Society, Cancer Facts and Figures, 2000, Atlanta,    Ga.-   4. Sherman et al. Cancer 84: 273-80, 1998.-   5. Brown et al. JAMA 281:347-53, 1999.-   6. Schechter, Acta Cytol. 40: 1272-82, 1996.-   7. Sawaya et al. Obstet Gynecol 94:307-10, 1999.-   8. Mandelblatt et al. JAMA 287:2372-81, 2002.-   9. Sherman et al. J Natl Cancer Inst 94:102-7, 2002.-   10. Vassilakos et al. Br J Cancer 86:382-8, 2002.-   11. Weaver et al. Acta Cytol 44:301-4, 2000.-   12. Lin, et al. Am J Obstet Gynecol 183:39-45, 2000.-   13. Patterson et al. Acta Cytol 45:36-47, 2001.-   14. Walboomers et al. J Pathol 189:12-9, 1999.-   15. Doeberitz, Pap Report 13: 65-74, 2002.-   16. Feichter et al., Acta Cytol 46:630-2, 2002.-   17. Jeon et al., J. Virol. 69(5):2989-2997, 1995.-   18. Bosch et al., J Clin Pathol 55:244-65, 2002.-   19. Brooks et al., Br J Cancer 86:263-8, 2002.-   20. Keesee et al. Anal Quant Cytol Histol 24:137-46, 2002.-   21. Bibbo et al. Acta Cytol 46: 25-9, 2002.-   22. Tjalma et al., European J of Obstet & Gyn and Reproductive    Biology 97: 223-230, 2001.-   23. Keating et al., The American J of Surgical Pathology 25: 884-91,    2001.-   24. Keating et al., Advances in Anatomic Pathology, 8: 83-92, 2001.-   25. Sano et al., Pathology International 52: 375-83, 2002.

1. A method of determining a neoplastic risk status of a subject,comprising: (a) measuring HPV DNA levels from a sample collected from asubject, wherein an HPV DNA level signal to cutoff ratio of greater thanor equal to 1 is indicative of HPV-infection and risk of neoplasia; (b)measuring protein marker expression levels from the sample collectedfrom the subject of step (a) using at least one protein marker selectedfrom the group consisting of: a cell proliferation group and cell cyclecontrol group protein, wherein a protein marker expression level signalto cutoff ratio of greater than or equal to 1 is indicative of proteinmarker expression; and (c) determining the neoplastic risk status of thesubject.
 2. A method of determining a neoplastic risk status of asubject, comprising: (a) measuring HPV RNA levels from a samplecollected from a subject, wherein an HPV RNA level signal to cutoffratio of greater than or equal to 1 is indicative of HPV-infection andrisk of neoplasia; (b) measuring protein marker expression levels fromthe sample collected from the subject of step (a) using at least oneprotein marker selected from the group consisting of: a cellproliferation group and cell cycle control group protein, wherein aprotein marker expression level signal to cutoff ratio of greater thanor equal to 1 is indicative of protein marker expression; and (c)determining the neoplastic risk status of the subject.
 3. The method ofclaim 1 or 2, wherein the subject sample does not have high risk HPVnucleic acids and does not have protein marker expression, said subjecthas a neoplastic risk status of Molecular Grade
 0. 4. The method ofclaim 1 or 2, wherein the subject sample has high risk HPV nucleic acidsand does not have protein marker expression, said subject has aneoplastic risk status of Molecular Grade I.
 5. The method of claim 1 or2, wherein the subject sample has high risk HPV nucleic acids andprotein marker expression, said subject has a neoplastic risk status ofMolecular Grade II.
 6. The method of claim 1 or 2, wherein the proteinexpression levels are measured by immunoassay.
 7. The method of claim 1or 2, wherein the immunoassay is selected from the group consisting of:ELISA, LUMINEX- and immunocytochemistry-based assays, and protein microarray.
 8. The method of claim 7, wherein the ELISA detects proteinexpression by a detection means selected from the group consisting of:radioactivity, color, chemiluminescence, and fluorescence.
 9. The methodof claim 1 or 2, wherein the cell proliferation group protein marker isselected from the group consisting of: PCNA, MIB-1, Ki-67, cdc6, mcm2,and mcm5.
 10. The method of claim 1 or 2, wherein the cell cycle controlgroup protein marker is selected from the group consisting of: p16, p14,and p21.
 11. The method of claim 1 or 2, wherein the protein marker isselected from the group consisting of: PCNA and p16.
 12. The method ofclaim 1 or 2, wherein the protein marker is PCNA.
 13. A method ofcategorizing HPV-induced cervical neoplasia and cancer in a subject,comprising: (a) detecting a presence or an expression of HPV nucleicacid levels in a subject sample; (b) detecting a protein markerexpression in the subject sample, wherein at least one protein marker isselected from the group consisting of: PCNA, MIB-1, Ki-67, cdc6, mcm2,mcm5, p16, p14, and p21; (c) determining a Molecular Grade of thesubject sample, wherein Molecular Grade 0 is characterized by an absenceof high risk HPV DNA or RNA; Molecular Grade I is characterized by apresence of high-risk HPV DNA or RNA and an absence of protein markerexpression, and Molecular Grade II is characterized by a presence ofhigh risk HPV DNA or RNA and a presence of protein marker expression;and (d) categorizing the HPV-induced cervical neoplasia and cancer intoMolecular Grade 0, Molecular Grade I, or Molecular Grade II in thesubject.
 14. The method of claim 13, wherein the subject sample isMolecular Grade 0 for: 0-18% of a population having high grade squamousintraepithelial lesion (HSIL; suspect cervical intraepithelial neoplasiaCIN 3+); 1-12% of a population having high grade squamousintraepithelial lesion (HSIL; suspect CIN2-3); 8-23% of a populationhaving low grade squamous intraepithelial lesion (LSIL; suspect CIN 1);41-63% of a population having atypical squamous cells of undeterminedsignificance (ASCUS); and 85-97% of a population within normal limits(WNL).
 15. The method of claim 13, wherein the subject sample isMolecular Grade I for: 0-18% of a population having HSIL (suspectCIN3+); 6-21% of a population having HSIL (suspect CIN2-3); 28-49% of apopulation having LSIL (suspect CIN1); 12-29% of a population havingASCUS; and 2-11% of a population within normal limits.
 16. The method ofclaim 13, wherein the subject sample is Molecular Grade II for: 82-100%of a population having HSIL (suspect CIN3+); 74-91% of a populationhaving HSIL (suspect CIN2-3); 37-59% of a population having LSIL(suspect CIN1); 19-39% of a population having ASCUS; and 0-8% of apopulation within normal limits.
 17. A method of reducing a number offalse positive results in determining a neoplastic risk status of asubject, comprising: (a) measuring a presence or expression of HPVnucleic acid levels from a sample collected from a subject, wherein anHPV nucleic acid level signal to cutoff ratio greater than or equal to 1is indicative of HPV infection, risk of neoplasia, and the presence orexpression of high risk HPV nucleic acids; and (b) measuring proteinmarker expression levels from the sample collected from the subject ofstep (a) using at least one protein marker selected from the groupconsisting of: a cell proliferation group and cell cycle control groupprotein, wherein a protein marker expression level signal to cutoffratio greater than or equal to 1 is indicative of protein markerexpression; thereby, reducing the number of false positive results indetermining the neoplastic risk status of the subject infected with HPV.18. The method of claim 17, wherein the number of false positive resultsis reduced by 15% to 100%.
 19. The method of claim 17, wherein thenumber of false positive results is reduced by 20% to 90%.
 20. Themethod of claim 17, wherein the number of false positive results isreduced by 30%-70%.
 21. A method of determining a neoplastic risk statusof a subject, comprising: (a) measuring a presence or expression of HPVnucleic acid levels from a sample collected from a subject, wherein anHPV nucleic acid level signal to cutoff ratio of greater than or equalto 1 is indicative of HPV-infection, risk of neoplasia, and the presenceor expression of high risk HPV nucleic acids, wherein the method ofmeasuring HPV nucleic acid levels uses low stringency conditions; (b)measuring protein marker expression levels from the sample collectedfrom the subject of step (a) using at least one protein marker selectedfrom the group consisting of: a cell proliferation group and cell cyclecontrol group protein, wherein a protein marker expression level signalto cutoff ratio of greater than or equal to 1 is indicative of proteinmarker expression; and (c) determining the neoplastic risk status of thesubject.
 22. A method of determining a neoplastic risk status of asubject, comprising: (a) measuring a presence or expression of HPVnucleic acid levels from a sample collected from a subject, wherein anHPV nucleic acid level signal to cutoff ratio of greater than or equalto 1 is indicative of HPV-infection, risk of neoplasia, and the presenceor expression of high risk HPV nucleic acids, wherein the method ofmeasuring HPV nucleic acid levels uses high stringency conditions; (b)measuring protein marker expression levels from the sample collectedfrom the subject of step (a) using at least one protein marker selectedfrom the group consisting of: a cell proliferation group and cell cyclecontrol group protein, wherein a protein marker expression level signalto cutoff ratio of greater than or equal to 1 is indicative of proteinmarker expression; and (c) determining the neoplastic risk status of thesubject.
 23. The method of any one of claims 13, 17, 21, and 22, whereinthe HPV nucleic acid is selected from the group consisting of: DNA andRNA.
 24. The method of any one of claims 21 and 22, wherein the methodof measuring the presence or expression of HPV nucleic acid levels isselected from the group consisting of: polymerase chain reaction,Southern blot, in situ hybridization, branched DNA assays,transcription-mediated amplification, ligase chain reaction,self-sustained sequence replication, nucleic acid sequence basedamplification, strand displacement amplification, and amplification withQβ replicase.
 25. A kit for detecting high risk HPV nucleic acids andsubsequently detecting protein marker expression in a sample,comprising: at least two probes capable of detecting high risk HPVnucleic acids in a sample, and at least one primary antibody that isspecific to a protein marker selected from the group consisting of:PCNA, Ki-67, cdc6, mcm2, mcm5, p16, p14, and p21, wherein the two probesare useful for detecting the high risk HPV nucleic acids and the primaryantibody is useful for detecting the corresponding protein markers inthe sample.
 26. The kit according to claim 25, wherein at least oneprobe is selected from the group consisting of: a DNA probe and an RNAprobe.
 27. A method of determining a neoplastic risk status of asubject, comprising: (a) measuring HPV DNA levels from a samplecollected from a subject, wherein an HPV DNA level signal to cutoffratio of greater than or equal to 1 is indicative of HPV-infection andrisk of neoplasia; (b) measuring protein marker expression levels fromthe sample collected from the subject of step (a) using one cellproliferation group protein marker and one cell cycle control groupprotein marker, wherein a protein marker expression level signal tocutoff ratio of greater than or equal to 1 is indicative of proteinmarker expression; and (c) determining the neoplastic risk status of thesubject.
 28. A method of determining a neoplastic risk status of asubject, comprising: (a) measuring HPV RNA levels from a samplecollected from a subject, wherein an HPV RNA level signal to cutoffratio of greater than or equal to 1 is indicative of HPV-infection andrisk of neoplasia; (b) measuring protein marker expression levels fromthe sample collected from the subject of step (a) using one cellproliferation group protein marker and one cell cycle control groupprotein marker, wherein a protein marker expression level signal tocutoff ratio of greater than or equal to 1 is indicative of proteinmarker expression; and (c) determining the neoplastic risk status of thesubject.
 29. The method of claim 27 or 28, wherein the subject sampledoes not have high risk HPV nucleic acids and does not have proteinmarker expression, said subject has a neoplastic risk status ofMolecular Grade
 0. 30. The method of claim 27 or 28, wherein the subjectsample has high risk HPV nucleic acids and does not have protein markerexpression, said subject has a neoplastic risk status of Molecular GradeI.
 31. The method of claim 27 or 28, wherein the subject sample has highrisk HPV nucleic acids and protein marker expression, said subject has aneoplastic risk status of Molecular Grade II.
 32. The method of claim 27or 28, wherein the protein expression levels are measured byimmunoassay.
 33. The method of claim 27 or 28, wherein the immunoassayis selected from the group consisting of: ELISA, LUMINEX- andimmunocytochemistry-based assays, and protein micro array.
 34. Themethod of claim 33, wherein the ELISA detects protein expression by adetection means selected from the group consisting of: radioactivity,color, chemiluminescence, and fluorescence.
 35. The method of claim 27or 28, wherein the cell proliferation group protein marker is selectedfrom the group consisting of: PCNA, MIB-1, Ki-67, cdc6, mcm2, and mcm5.36. The method of claim 27 or 28, wherein the cell cycle control groupprotein marker is selected from the group consisting of: p16, p14, andp21.
 37. The method of claim 27 or 28, wherein the cell proliferationgroup protein marker is PCNA and the cell cycle control group proteinmarker is p16.
 38. A method of categorizing HPV-induced cervicalneoplasia and cancer in a subject, comprising: (a) detecting a presenceor an expression of HPV nucleic acid levels in a subject sample; (b)detecting a protein marker expression in the subject sample, wherein oneprotein marker is selected from the group consisting of: PCNA, MIB-1,Ki-67, cdc6, mcm2, and mcm5; (c) detecting a protein marker expressionin the subject sample, wherein one protein marker is selected from thegroup consisting of: p16, p14, and p21; (d) determining a MolecularGrade of the subject sample, wherein Molecular Grade 0 is characterizedby an absence of high risk HPV DNA or RNA; Molecular Grade I ischaracterized by a presence of high-risk HPV DNA or RNA and an absenceof protein marker expression, and Molecular Grade II is characterized bya presence of high risk HPV DNA or RNA and a presence of protein markerexpression; and (e) categorizing the HPV-induced cervical neoplasia andcancer into Molecular Grade 0, Molecular Grade I, or Molecular Grade IIin the subject.
 39. The method of claim 38, wherein the subject sampleis Molecular Grade 0 for: 0-18% of a population having high gradesquamous intraepithelial lesion (HSIL; suspect cervical intraepithelialneoplasia CIN 3+); 1-12% of a population having high grade squamousintraepithelial lesion (HSIL; suspect CIN2-3); 8-23% of a populationhaving low grade squamous intraepithelial lesion (LSIL; suspect CIN 1);41-63% of a population having atypical squamous cells of undeterminedsignificance (ASCUS); and 85-97% of a population within normal limits(WNL).
 40. The method of claim 38, wherein the subject sample isMolecular Grade I for: 0-18% of a population having HSIL (suspectCIN3+); 6-21% of a population having HSIL (suspect CIN2-3); 28-49% of apopulation having LSIL (suspect CIN1); 12-29% of a population havingASCUS; and 2-11% of a population within normal limits.
 41. The method ofclaim 38, wherein the subject sample is Molecular Grade II for: 82-100%of a population having HSIL (suspect CIN3+); 74-91% of a populationhaving HSIL (suspect CIN2-3); 37-59% of a population having LSIL(suspect CIN1); 19-39% of a population having ASCUS; and 0-8% of apopulation within normal limits.
 42. A method of reducing a number offalse positive results in determining a neoplastic risk status of asubject, comprising: (a) measuring a presence or expression of HPVnucleic acid levels from a sample collected from a subject, wherein anHPV nucleic acid level signal to cutoff ratio greater than or equal to 1is indicative of HPV infection, risk of neoplasia, and the presence orexpression of high risk HPV nucleic acids; and (b) measuring proteinmarker expression levels from the sample collected from the subject ofstep (a) using one cell proliferation group protein marker and one cellcycle control group protein marker, wherein a protein marker expressionlevel signal to cutoff ratio greater than or equal to 1 is indicative ofprotein marker expression; thereby, reducing the number of falsepositive results in determining the neoplastic risk status of thesubject infected with HPV.
 43. The method of claim 42, wherein thenumber of false positive results is reduced by 15% to 100%.
 44. Themethod of claim 42, wherein the number of false positive results isreduced by 20% to 90%.
 45. The method of claim 42, wherein the number offalse positive results is reduced by 30%-70%.
 46. A method ofdetermining a neoplastic risk status of a subject, comprising: (a)measuring a presence or expression of HPV nucleic acid levels from asample collected from a subject, wherein an HPV nucleic acid levelsignal to cutoff ratio of greater than or equal to 1 is indicative ofHPV-infection, risk of neoplasia, and the presence or expression of highrisk HPV nucleic acids, wherein the method of measuring HPV nucleic acidlevels uses low stringency conditions; (b) measuring protein markerexpression levels from the sample collected from the subject of step (a)using one cell proliferation group protein marker and one cell cyclecontrol group protein marker, wherein a protein marker expression levelsignal to cutoff ratio of greater than or equal to 1 is indicative ofprotein marker expression; and (c) determining the neoplastic riskstatus of the subject.
 47. A method of determining a neoplastic riskstatus of a subject, comprising: (a) measuring a presence or expressionof HPV nucleic acid levels from a sample collected from a subject,wherein an HPV nucleic acid level signal to cutoff ratio of greater thanor equal to 1 is indicative of HPV-infection, risk of neoplasia, and thepresence or expression of high risk HPV nucleic acids, wherein themethod of measuring HPV nucleic acid levels uses high stringencyconditions; (b) measuring protein marker expression levels from thesample collected from the subject of step (a) using one cellproliferation group protein marker and one cell cycle control groupprotein marker, wherein a protein marker expression level signal tocutoff ratio of greater than or equal to 1 is indicative of proteinmarker expression; and (c) determining the neoplastic risk status of thesubject.
 48. The method of any one of claims 38, 42, 46, and 47, whereinthe HPV nucleic acid is selected from the group consisting of: DNA andRNA.
 49. The method of any one of claims 46 and 47, wherein the methodof measuring the presence or expression of HPV nucleic acid levels isselected from the group consisting of: polymerase chain reaction,Southern blot, in situ hybridization, branched DNA assays,transcription-mediated amplification, ligase chain reaction,self-sustained sequence replication, nucleic acid sequence basedamplification, strand displacement amplification, and amplification withQβ replicase.